Background
DN is the largest single cause of ESRD in many developed countries and will continue to be the leading cause of death in diabetes mellitus. DN occurs in 20% to 40% of all patients with type 2 diabetes mellitus, accounting for approximately 50% of cases in the developed countries.[1] The costs of care for people with DKD are extraordinarily high. In the Medicare population alone, DKD-related expenditures among this mostly older group were nearly $25 billion in 2011.[2] In China, the incidence of diabetic nephropathy is also increasing at an alarming rate due to the rapid increase of diabetes prevalence and low awareness rate. As the initial symptoms of chronic nephropathy are very secretive, hypertension, urinary protein, edema and other obvious symptoms have entered the stage of clinical diabetic nephropathy or dominant diabetic nephropathy (overtDN). Furthermore, patients often fail to go to the hospital for examination, so patients with diabetic nephropathy often miss the optimal intervention period. Failure to intervene early can lead to a dramatic deterioration in kidney function and a 90 per cent mortality rate. However, if the renal status of patients can be monitored at any time, the reversion rate can be as high as 70% if intervention is carried out before stage iii of diabetic nephropathy (incipientDN).
At present, urinary microalbumin (mALB) excretion rate is mainly used to diagnose diabetic nephropathy. Although the detection method is relatively perfect, it has the following disadvantages:
- The accuracy and specificity of mALB in diagnosing DN are not high, and factors such as urinary tract infection can increase the excretion rate of urinary albumin, leading to misdiagnosis and missed diagnosis.
- When mALB is abnormal, DN has progressed to stage iii, and at this time, the patient's GBM thickening and mesangial matrix increase are more obvious, and there are already glomerular nodules and diffuse lesions as well as small artery hyaline changes, and the glomeruli have become dilapidated.
Han-Henrik Parving said, "Microalbuminuria may not be as sensitive and specific a predictor of diabetic nephropathy as previously suggested. Other markers of risk for diabetic nephropathy are needed for optimal clinical management."[3] Studies have shown that the urine of diabetic nephropathy patients with beta2 microglobulin was abnormal 5 years earlier than that of urinary microalbumin.[4]
Our social practice survey found that people are much more receptive to urine tests than blood tests. So our project was launched to provide diabetes patients with a home, visual, portable test box for early and early kidney disease using beta2 microglobulin as a marker.
Principle brief introduction
In this project, we combined the highly specific nanobody with the highly sensitive liquid crystal detection to develop a detection method that meets the above conditions, and we hope to achieve the semi-quantitative visual analysis of ß2m in urine of 150-400ng/ml. The normal concentration of ß2m in human urine is about 150ng/ml, and other obvious symptoms appear when the concentration of buxi2m in urine is about 450ng/ml. Firstly, nanobody was selected to specifically bind the ß2m in urine, and long chain modification was carried out for improving detection sensitivity. We performed the sentinel modification of the nanobacteria using a method catalyzed by enterobacterium, and then linked other molecules with active and specialized aldehyde groups. Compared with traditional antibodies, nanobody has smaller molecular weight, is not easily denatured, and still has complete antigen-binding property. Moreover, the nanobody can be expressed by escherichia coli as the host, and the preparation cost is greatly reduced.
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Fig.1 A schematic of catalysis in E. coli |
More subtly, we took advantage of crystals' fluidity and anisotropy. First, the substrate of the liquid crystal pool was modified with an antigen, and then a certain concentration of nanobody and the mixture of the solution to be tested were added. After a period of reaction, some antibodies preferentially bound with the scaffold ß2M in the solution, and the other with the substrate ß2M. After the glass slides were rinsed, the microenvironment of the liquid crystal base changed due to the combination of ß2m and Nb, resulting in obvious color or light and shade differences of the liquid crystal, thus realizing the visualization of detection. Finally, we expressed the brightness of the liquid crystal with the gray value of the image, and established the relationship between the fixed antigen concentration of the liquid crystal substrate and the gray value of the image.
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Fig.2 The object to be tested changes the orientations of the liquid crystal alignment |
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Results and outlook
Based on the above principles and design, we firstly verified that there was a significant correlation between the liquid crystal brightness and the concentration of fixed mountain 2M, and through the optimization of the parameters of the detection system, the liquid crystal brightness changed significantly when the antigen concentration was 150ng/mL-400ng/mL. In addition, the antibody with long chain modification is selected for detection, and the brightness changes of the liquid crystal are more obvious, which can achieve the purpose of effective and reliable detection.
At the same time, we designed a test box of 5cm × 5cm × 10cm to demonstrate our product. The short-term goal of this project is to make the patients in the home use of our test kits, only need to add a drop of urine and auxiliary drops wait for a period of time after taking a picture, can intuitive understanding to your beta 2 m concentration in the urine and kidney function, when he will photos uploaded to the mobile terminal, can generate individualized treatment guidelines.
Of course, we have a better idea. We can use this test to determine a variety of proteins. After all, there are many diseases that require more accurate diagnosis using one or a series of proteins in urine or blood. For example, AD7c-ntp in urine can be used as an indicator of alzheimer's disease. The index of coronary heart disease is 15 kinds of urine polypeptide combination. To do this we simply need to design a new type of 96-well plate that imparts different antigens to the substrate to enable the determination of multiple proteins and even proteomics.
[1] Xue R, Gui D, Zheng L, Zhai R, Wang F, Wang N. Mechanistic Insight and Management of Diabetic Nephropathy: Recent Progress and Future Perspective. Journal of Diabetes Research. 2017; 2017:1839809.
[2] Tuttle KR, Bakris GL, Bilous RW, et al. Diabetic Kidney Disease: A Report From an ADA Consensus Conference. Diabetes Care. 2014; 37(10):2864-2883.
[3] Han-Henrik Parving, Nish Chaturvedi, GianCarlo Viberti, Carl Erik Mogensen. Does Microalbuminuria Predict Diabetic Nephropathy? , Diabetes Care Feb, 2002, 25 (2) 406-40
[4] Tóth L, Szénási P, Varsányi NM, Szilvási I, Kammerer L. Clinical significance of beta-2-microglobulin in diabetes mellitus, Orv Hetil. 1989; 130(5):223-5.
